(a) Field of Invention
This invention relates to peptides of the formula A-B-C (1) and pharmaceutically acceptable salts thereof in which A is selected from the group consisting of L-pyroglutamyl (L-H-(pyro)-Glu), D-pyroglutamyl (D-H-(pyro)-Glu), and L-homo-pyroglutamyl (L-H-homo-(pyro)-Glu); B is selected from the group consisting of L-histidyl (L-His), D-histidyl (D-His), L-3'-methylhistidyl (L-N.sup.im -3-MeHis), L-phenylalanyl (L-Phe), L-p-aminophenylalanyl(L-p-NH.sub.2 Phe), and L-.beta.-(pyrazolyl-1)alanyl (L-.beta.-(pyrazolyl-1)Ala); and C is selected from the group consisting of glycine and lower alkyl esters thereof (Gly-OR in which R is H or lower alkyl), glycine amide and lower alkyl amides thereof (Gly-NHR in which R is H or lower alkyl), 2-amino-1-hydroxyethyl (GLy-ol), D-alanine (D-Ala), L-.beta.-(2-thienyl)alanine, and NHR.sup.1 in which R.sup.1 is lower alkyl, with the proviso that C may not be glycine or glycinamide when A is L-pyroglutamyl and B is L-histidyl. This invention also relates to a process for preparing said compounds of formula 1, to intermediates used in said process, and to the use of said compounds in mammals as anorexigenic agents in methods of treatment of obesity and of other pathological conditions in which a reduction of food intake is indicated, as well as to their use in methods of treatment of pathological states associated with excessive secretion of gastric acid or of pancreatic fluid such as gastric or duodenal ulcers or acute pancreatitis, and in certain critical states of the central nervous system such as reduced consciousness or coma due to brain injury.
The abbreviations used throughout this application for the amino acids or the residues thereof as well as for the protective groups are generally based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature, see Biochemistry 11, 1726 (1972), for example: (pyro)-Glu, 5-oxoproline or pyroglutamic acid; homo-(pyro)-Glu, homopyroglutamic acid; His, histidine; N.sup.im -3-MeHis, 3'-methylhistidine; Phe, phenylalanine; p-NH.sub.2 Phe, p-aminophenylalanine, .beta.-(pyrazolyl-1)Ala, .beta.-(pyrazol-1-yl)-alanine; Gly, glycine, Gly-ol, 2-aminoethanol; and Thi, .beta.-(2-thienyl) alanine. All amino acids have the natural or L-configuration unless stated otherwise; D-His is the residue of D-histidine, D-(pyro)-Glu is the residue of D-pyroglutamic acid, and D-Ala is the residue of D-alanine. The abbreviations Me and Et are used for methyl and ethyl, respectively. The abbreviations used for the protective groups are, for example: Boc, t-butyloxycarbonyl; Z, benzyloxycarbonyl; Tos, tosyl; Dnp, dinitrophenyl; N.sup.im means the imidazole nitrogen of histidine; and Y is a suitable anchoring group used in solid phase synthesis linked to a solid resin support, preferably ##STR1##
(b) Background of the Invention
The humoral control of appetite, especially via the hypothalamus, has been under discussion for many years, see e.g. the review by Schally et al., Am. J. Med. Sci. 248, (1), 79 (1964) and references cited therein. It was known at that time that the hypothalamus was involved in the regulation of food intake. Electrical stimulation of the lateral hypothalamic area produces hyperphagia and its destruction leads to aphagia. Contrarywise, the stimulation of the ventromedial nucleus decreased food intake, and its destruction leads to hyperphagia and obesity.
The obesity syndrome which follows the administration of gold thioglucose is in part the result of hyperphagia associated with hypothalamic lesions produced by this agent. Theories on the neural control of hunger, appetite and satiety have been ably reviewed by others and several mechanism have been suggested: (1) Thermostatic theory. According to this theory, specific dynamic action of food and its effect on body temperature regulate appetite. (2) Chemostatic theories. These postulate that appetite is regulated by intracellular or extracellular levels of glucose (glucostatic theory), lipids, (lipostatic theory, and also by the concentration of certain metabolites such as serum amino acids. (3) Another group of theories assert that sensations from the digestive tract associated with eating and presence of food in the stomach and the intestine regulate appetite. Among factors involved in the production of satiety or cessation of eating is the distention of stomach by food or non-nutritious substances. Gastric contractions may be related to hunger behavior, but these contractions are the same in animals rendered aphagic or hyperphagic by hypothalamic injury as in normal animals deprived of food and water.
These theories do not explain all the experimental facts, among them that the diabetic animal can be hungry. Schally et al. cited above then suggest that the obesity seen in animals with hypothalamic lesions could be mediated by a humoral rather than a neurogenic mechanism and that the hypothalamus may elaborate a substance involved in a central control of appetite. Preliminary evidence is cited which suggests the presence in neurohypophyseal extract of a substance which will affect the dynamic phase of weight gain in gold thioglucose-treated mice.
A more recent brief review of the status of investigation concerning hypothalamic control of food intake and obesity is found in Schally et al. Recent Progress in Hormone Research, Proceedings of the 1967 Laurentian Hormone Conference, 24, 497 (1968), in particular pp. 570-571 and references cited therein. Among those references is the paper by Schally et al, in Science 157, 210 (1967) which showed that administration of enterogastrone purified from hog duodenum reduced the food intake of mice fasted for 17 hours. This effect was greatest during the first 30 minutes, but the cumulative reduction continued for at least 4 hours. Other peptides prepared from hog duodenum or colon, as well as glucagon, secretin, glucose, and bovine serum albumin were ineffective. The authors state that this effect could have been due to a direct elimination of gastric hunger contractions or could have been mediated by an action through the central nervous system involving release of hypothalamic substances, although positive evidence for any hypothalamic neurohumors responsible for the indirect or direct control of appetite was still lacking at that time.
It would appear that this positive evidence was supplied by Tyrgstad et al., Acta Endocrinologica 89, 196 (1978) who isolated a number of peptides which produced metabolic behavioural effects from the urine of patients suffering from the hypothalamic syndrome congenital, general lipodistrophy. Anorexia nervosa is associated with hypothalamic disturbances. In primary hypothalamic anorexia nervosa, the hypothalamic-pituitary axis is disturbed, resulting in low release of gonadotropins, amenorrhea, loss of diurnal rhythm for ACTH secretion, reduced secretion of thyrotropin, and initial increase and later decrease in secretion of somatotropin.
Precipitates from urine specimens from 25 patients diagnosed as anorexia nervosa were chromatographed on Sephadex G-25 gel columns, and could be divided into four different patterns: One was similar to that for normal controls, one similar to that observed for patients with schizophrenia, 5 patients with a hysteriform type of neurosis had a third form of pattern, and 10 girls considered to have a primary "hypothalamic" type of anorexia nervosa also had typical chromatograms. Fractions influencing appetite in mice were found in the latter group only. Two peptides influencing appetite were purified through several steps of chromatography. One increased appetite and one depressed it in mice into which the peptides were injected. The peptides are enveloped in peptide-carrier proteins and are thus protected against enzymatic degradation, which made their isolation from urine possible.
The structure of the anorexigenic peptide was elucidated by Reichelt et al., Neuroscience 3, 1207 (1978) who worked in close association with Trygstad cited above, and it was determined to be the tripeptide H-(pyro)-Glu-His-GlyOH; this was confirmed by synthesis.
A dose of 12 nmole of appetite-retarding peptide injected in mice daily for 20 days reduced food consumption from 5.7 to 3.0 g per day for about 6 months. Body weight dropped from a mean of 35 g to a minimum of 24 g.
The tripeptide had no acute effect on blood glucose or serum insulin levels and seemed to act on receptors localized in the hypothalamic centers controlling appetite.
The appetite-stimulating peptide increased daily consumption of food to more than 10 g, and mean body weight jumped to 57 g. The structure of the appetite-stimulating peptide could not be identified.
Two similar factors inducing increased and decreased feeding behavior were also isolated from patients with genetic metabolic obesity.
We have found that peptides of the formula (1) A-B-C in which A, B, and C are as defined above are more active and have a longer duration of activity in reducing appetite and inhibiting food intake than the tripeptide pryoglutamyl-histidyl-glycine (H-(pyro)-Glu-His-Gly-OH) isolated from urine by Trygstad et al., and by Reichelt et al., both cited above. Those peptides of formula (1) and their pharmaceutically acceptable salts which are biologically equivalent to the above peptides themselves, are therefore useful as anorexiant agents in the treatment of obesity and of associated pathological conditions which require a reduction in food intake. They also reduce gastric and pancreatic secretion and are thus useful in the treatment of pathological conditions associated with excessive production of gastric acid and/or of pancreatic fluid. Moreover, they exert certain activities upon the central nervous system which makes them useful in the acute treatment of states of reduced consciousness.
They possess the advantages over the natural tripeptide of being more active and of having a longer duration of activity and both those attributes are of practical significance: the lesser minimum effective doses reducing side effects as well as the cost for the preparation of the compounds and the longer acting properties reducing the need for frequent administration.
In view of the biological equivalence of the peptides of formual 1 and of their pharmaceutically acceptable salts, all preceding and subsequent references to the peptides of formula 1 are to be understood as covering both said peptides and said salts.